The present invention relates to liquefying alkaline xcex1-amylases having high chelating-agent-resisting performance and are useful as a component for detergents.
xcex1-Amylases have been used widely in various industrial fields such as starch, brewing, fiber, pharmaceutical and food industries. Since they are known to be suited as one of the components of a detergent, they are incorporated even in an automatic dish washing detergent or a laundry detergent as a detergency reinforcing component (Enzymes in Detergency, p203, Marcel Dekker Inc., New York (1995)).
As liquefying xcex1-amylases useful for a detergent and having the optimum effects on the alkaline side, those previously found by the present inventors and derived from the strain Bacillus sp. KSM-1378 (FERM BP-3048) are known. Recently, xcex1-amylases having the optimum pH at around 8 to 9.5 are disclosed (WO95/2639). They resemble closely to those derived from the strain KSM-1378 in properties and structure.
In a detergent, a chelating agent such as phosphoric acid, citric acid or zeolite is incorporated to remove, from a washing liquid, cleansing-disturbing ions such as calcium ions. It has been known for long years that liquefying xcex1-amylases require calcium ions for expressing their enzyme activity but such calcium ions are deactivated by the above-described chelating agent or a stronger chelating agent EDTA [HANDBOOK OF AMYLASES AND RELATED ENZYMES, p43, The Amylase Research Society Japan(1988)]. In recent days, it is reported that X-ray crystallographic analysis of the liquefying xcex1-amylases known to date reveals that three calcium atoms exist in the molecule thereof and 13 amino acid residues are conserved with markedly high frequency [Structure, 6, 281(1998)].
Inhibition of enzyme activity by a chelating agent is also recognized in the above-described liquefying alkaline xcex1-amylase derived from the strain Bacillus sp. KSM-1378 (FERM BP-3048) and sufficient effects of this xcex1-amylase are not always exhibited when it is incorporated in an automatic dish washing detergent or laundry detergent. Liquefying xcex1-amylases (Termamyl and Duramyl, products of Novo Nordisk A/S) derived from Bacillus licheniformis, which are most frequently employed as a component of an automatic dish washing detergent or laundry detergent, are also insufficient in chelating-agent-resisting performance.
Among the liquefying amylases known to date, a liquefying xcex1-amylase (WO90/11352) derived from the strain belonging to Pyrococcus sp. and an xcex1-amylase (WO96/02633) which is derived from the strain belonging to Sulfolobus sp. and is effective in the liquefying step of a starch are free from the influence from a chelating agent. These enzymes however have the optimum acting pH in a range of 4 to 6 and 2.5 to 4.5, respectively and do not act in the alkaline range so that they are not suited as a component of a detergent.
An object of the present invention is therefore to provide a liquefying alkaline xcex1-amylase having higher chelating-agent-resisting performance than conventional amylases for a detergent and is useful as a component of a detergent; and a detergent composition having this liquefying alkaline xcex1-amylase incorporated therein.
In one aspect of the present invention, there is thus provided a liquefying alkaline amylase having residual activity not less than 70% when treated at pH 10 and 45xc2x0 C. for 30 minutes in the presence of 1 to 100 mM of EDTA or EGTA.
In another aspect of the present invention, there is also provided a DNA fragment encoding said liquefying alkaline amylase.
In a further aspect of the present invention, there is also provided a detergent composition containing said liquefying alkaline amylase.